{"title":"A comparison of SWATH-MS methods for measurement of residual host cell proteins in adeno-associated virus preparations.","authors":"Thomas M Leibiger, Lie Min, Kelvin H Lee","doi":"10.3389/fbioe.2025.1579098","DOIUrl":null,"url":null,"abstract":"<p><strong>Introduction: </strong>Analysis of residual host cell proteins in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. One strategy to address these challenges is through development of improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with greater sensitivity and reduced sample requirement.</p><p><strong>Methods: </strong>In this work, we compare the performance of four sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) methods for identification and quantitation of residual HCPs in rAAV2, -5, -8, and -9 preparations produced with human embryonic kidney 293 (HEK293) cells and purified using immunoaffinity chromatography. Key SWATH-MS parameters including spectral library construction (data dependent vs. <i>in silico</i>), data processing software (DIA-NN vs. Skyline), and mass spectrometer instrument (Sciex TripleTOF 6600 vs. Sciex ZenoTOF 7600) were assessed. Method attributes including sample requirement and processing time, and method outputs including protein and precursor identifications, host cell protein quantitation comparisons across methods, and quantitation coefficients of variance (CV) were considered to help establish a SWATH-MS workflow well-suited for rAAV HCP analytics.</p><p><strong>Results: </strong>A 78% increase in HCP identifications, 80% reduction in sample requirement, and 70% reduction in instrument runtime was achieved with an <i>in silico</i> spectral library, data processing in DIA-NN, and data collection with the Sciex ZenoTOF 7600 instrument (DIA-NN-7600 method) compared to a previously established method using a DDA-derived spectral library, data processing in Skyline, and data collection with the Sciex TripleTOF 6600 instrument (Skyline-DDA-6600 method). Additionally, the DIA-NN-7600 method shows median HCP quantitation CV below 10% for triplicate data acquisitions, and comparable quantitation to other methods for a panel of highly abundant residual HCPs previously identified in rAAV downstream processing.</p><p><strong>Discussion: </strong>This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.</p>","PeriodicalId":12444,"journal":{"name":"Frontiers in Bioengineering and Biotechnology","volume":"13 ","pages":"1579098"},"PeriodicalIF":4.3000,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081442/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Bioengineering and Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.3389/fbioe.2025.1579098","RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Introduction: Analysis of residual host cell proteins in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. One strategy to address these challenges is through development of improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with greater sensitivity and reduced sample requirement.
Methods: In this work, we compare the performance of four sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) methods for identification and quantitation of residual HCPs in rAAV2, -5, -8, and -9 preparations produced with human embryonic kidney 293 (HEK293) cells and purified using immunoaffinity chromatography. Key SWATH-MS parameters including spectral library construction (data dependent vs. in silico), data processing software (DIA-NN vs. Skyline), and mass spectrometer instrument (Sciex TripleTOF 6600 vs. Sciex ZenoTOF 7600) were assessed. Method attributes including sample requirement and processing time, and method outputs including protein and precursor identifications, host cell protein quantitation comparisons across methods, and quantitation coefficients of variance (CV) were considered to help establish a SWATH-MS workflow well-suited for rAAV HCP analytics.
Results: A 78% increase in HCP identifications, 80% reduction in sample requirement, and 70% reduction in instrument runtime was achieved with an in silico spectral library, data processing in DIA-NN, and data collection with the Sciex ZenoTOF 7600 instrument (DIA-NN-7600 method) compared to a previously established method using a DDA-derived spectral library, data processing in Skyline, and data collection with the Sciex TripleTOF 6600 instrument (Skyline-DDA-6600 method). Additionally, the DIA-NN-7600 method shows median HCP quantitation CV below 10% for triplicate data acquisitions, and comparable quantitation to other methods for a panel of highly abundant residual HCPs previously identified in rAAV downstream processing.
Discussion: This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.
期刊介绍：
The translation of new discoveries in medicine to clinical routine has never been easy. During the second half of the last century, thanks to the progress in chemistry, biochemistry and pharmacology, we have seen the development and the application of a large number of drugs and devices aimed at the treatment of symptoms, blocking unwanted pathways and, in the case of infectious diseases, fighting the micro-organisms responsible. However, we are facing, today, a dramatic change in the therapeutic approach to pathologies and diseases. Indeed, the challenge of the present and the next decade is to fully restore the physiological status of the diseased organism and to completely regenerate tissue and organs when they are so seriously affected that treatments cannot be limited to the repression of symptoms or to the repair of damage. This is being made possible thanks to the major developments made in basic cell and molecular biology, including stem cell science, growth factor delivery, gene isolation and transfection, the advances in bioengineering and nanotechnology, including development of new biomaterials, biofabrication technologies and use of bioreactors, and the big improvements in diagnostic tools and imaging of cells, tissues and organs.
In today`s world, an enhancement of communication between multidisciplinary experts, together with the promotion of joint projects and close collaborations among scientists, engineers, industry people, regulatory agencies and physicians are absolute requirements for the success of any attempt to develop and clinically apply a new biological therapy or an innovative device involving the collective use of biomaterials, cells and/or bioactive molecules. “Frontiers in Bioengineering and Biotechnology” aspires to be a forum for all people involved in the process by bridging the gap too often existing between a discovery in the basic sciences and its clinical application.
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